1. Field of the Invention
The present invention relates to the quantitative determination of a particular nucleic acid segment in a sample. The invention is particularly useful for determining the quantity of specific mRNA molecules in a biological sample. The method is therefore especially applicable in the field of medical diagnostics but can also be applied in the fields of genetics, molecular biology, and biochemistry.
2. Description of Related Disclosures
U.S. Pat. Nos. 4,683,195 and 4,683,702 disclose methods for carrying out the polymerase chain reaction (PCR), a nucleic acid amplification method, and for using PCR in the detection of specific nucleotide sequences. European Patent Office Publication (EPO) No. 258,017 describes Taq polymerase, a preferred DNA polymerase for use in PCR. These publications are incorporated by reference herein.
PCR methods have widespread applications in genetic disease diagnosis (see Wu et al., 1989, Proc. Natl. Acad. Sci. USA 86:2757-2760 and Myerswitz, 1988, Proc. Natl. Acad. Sci. USA 85:3955-3959), as well as disease susceptibility and cancer diagnosis (see Horn et al., 1988, Proc. Natl. Acad. Sci. USA 85:6012-6016; Todd et al., 1987, Nature 329:599-604; Kawasaki, 1988, Proc. Natl. Acad. Sci. USA 5698-5702; and Neri et al., 1988, Proc. Natl. Acad. Sci. USA 85:9268-9272). However, these uses have provided only qualitative results by, for example, detecting unique mRNA transcripts from abnormal cells in a background of normal cells.
An attempt to use PCR for quantitative studies of mRNA levels for thymidylate synthase in tumors has been published (see Kashani-Sabet, 1988, Cancer Res. 48:5775-5778). However, this study provides only relative comparisons of amounts of mRNA in biological samples. It has been much more difficult to quantitate the absolute amount of specific mRNA without an internal standard of known concentration. Other methods have been described for quantitating nucleic acid species by using PCR to co-amplify a second, unrelated, template cDNA (see Chelly et al., 1988, Nature 333:858-860 and Rappolee et al., 1988, Science 241:708-712). The use of an unrelated cDNA standard also necessitates the use of a second set of oligonucleotide primers, unrelated to the specific target mRNA.
Because amplification is an exponential process, small differences in any of the variables which control the reaction rate, including the length and nucleotide sequence of the primer pairs, can lead to dramatic differences in the yield of PCR product. Analyses which use two sets of unrelated primers, therefore, can only provide a relative comparison of two independent amplification reactions rather than an absolute measure of mRNA concentration.
Gilliland et al. (J. Cellular Biochemistry, UCLA Symposia on Molecular and Cellular Biology, Apr. 3-21, 1989, Abstract WH001) describe alternative approaches to mRNA quantitation to avoid some of the problems associated with unrelated templates as amplification standards. However, the Gilliland et al. suggestions have other inherent limitations. One approach requires mapping of genomic introns and exons for the gene corresponding to a specific target mRNA. Gilliland et al. also proposes an alternative approach using site directed mutagenesis to construct an internal standard, which causes the formation of heteroduplexes following amplification. These heteroduplexes result in an over estimation of the amount of target sequence present in the original sample. Smith et al. (Smith et al., 1989, J. Immunol. Meth. 118:265-272) have used an RNA dot blot assay to assess quantitatively the expression level of the two IL-1 mRNAs in human macrophages. Smith et al. reported that the level of sensitivity for IL-1.alpha. mRNA was approximately 10.sup.7 molecules by his method, and IL-1.alpha. mRNA was undetected in uninduced macrophages. The present invention provides a quantitation method which can readily measure 10.sup.4 molecules and readily detects IL-1.alpha. mRNA in uninduced as well as induced macrophages in a sample assay. This 1000 fold increase in sensitivity represents an important advance in quantitative analysis for clinical and research purposes.
There remains a need for a method to quantitate directly, accurately, and reproducibly the amount of a specific nucleic acid segment in a sample. The availability of quantitative PCR will provide valuable information in a number of research areas. More particularly, the invention provides critical information in disease diagnosis and cancer therapy. For example, a reliable, sensitive, quantitative analysis can be critical in determining the extent of induction of mRNA synthesis in response to exogenous stimuli. The present invention overcomes the numerous limitations inherent in the attempts of others in this field, and thus provides means for accurately quantifying the amount of a nucleic acid segment in a biological sample.